Antibiotic resistance complicates the majority of Staphylococcus aureus (S. aureus) infections, as a full two thirds of hospital-associated S. aureus infections and ~50% of those acquired in the community are now methicillin-resistant (MRSA). MRSA causes >450,000 infections in the US each year, and it is responsible for half of all deaths caused by drug-resistant bacteria. The increasing incidence of multi-drug resistance in S. aureus and other bacteria underscores the need for next-generation antibiotics capable of combating these dangerous pathogens. The majority of small molecule antibiotics inhibit genetically-encoded intracellular enzymes, and as a result they are subject to rapid evolution of bacterial resistance. An alternative therapeutic strategy leverages recombinant enzymes, such as Staphylococcus simulans lysostaphin (ssLST), which degrade cell wall peptidoglycan causing bacterial lysis and death. Due to peptidoglycan's conserved nature and complex biosynthesis, such lytic enzymes have proven less susceptible to evolved resistance. Unfortunately, as a bacterial protein itself, ssLST is known to drive a potent immune response in animals and humans. This immunogenicity and associated toxicity represent critical barriers to ssLST clinical translation. This project seeks to design and develop immunotolerant ssLST drug candidates. We will employ cutting- edge computational deimmunization algorithms and advanced biomolecular engineering and immunogenicity screening technologies to identify and silence immunogenic T cell epitopes within the ssLST sequence. Importantly, our innovative methods simultaneously optimize protein therapeutics for both low immunogenic potential and high level function. We have previously deimmunized the ssLST catalytic domain, and here we aim to develop immunotolerant ssLST cell wall binding domains so as to complete global deimmunization of the protein. In Aim 1, proprietary deimmunization algorithms will be applied to design functionally deimmunized variants of the ssLST cell wall binding domain. In Aim 2, selected cell wall binding domain variants will be fused to our existing deimmunized catalytic domains, and the full length proteins will be characterized by analysis of expression yield, thermostability, bacterial lysis kinetics, and antibacterial activity as measured by minimal inhibitory concentration. Aim 3 will assess the immunogenicity of lead candidates using ex vivo immunoassays with human peripheral blood mononuclear cells. Specifically, immunoreactive T cells in donor samples will be quantified following stimulation with either wild type or deimmunized ssLST. The computational design expertise of Stealth Biologics LLC offers powerful synergy with the experimental capabilities of the Dartmouth research laboratories, and the proposed partnership is built upon a proven 7-year collaboration that has opened new frontiers in the field of biotherapeutic deimmunization. Successfully achieving the project goals will ultimately yield potent anti-staphylococcal drugs that have been optimized so as to provide safe and highly efficacious treatment of MRSA and other staph infection in humans.